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1- Making Density Gradients






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1- Making Density Gradients. Pre-formed discontinuous gradients Pre-formed continuous gradients Self-generated gradients. 2- Density Gradient Harvesting. C. A. B. Discontinuous gradient by overlayering. A. B. C. D. Discontinuous gradient by underlayering. density. top. bottom.
1- Making Density Gradients

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Slide 1

1- Making Density Gradients

  • Pre-formed discontinuous gradients

  • Pre-formed continuous gradients

  • Self-generated gradients

2- Density Gradient Harvesting

Slide 2

C

A

B

Discontinuous gradient by overlayering

Slide 3

A

B

C

D

Discontinuous gradient by underlayering

Slide 4

density

top

bottom

Diffusion of a discontinuous gradient

Slide 5

Discontinuous

Continuous

45-60 min

Rapid formation of continuous from discontinuous gradient

Slide 6

A

B

T

SB

SB

P

M

Two-chamber device for continuous gradients

Slide 7

B

The Gradient Master

Slide 8

Density (g/ml)

2 min

1.13

2.5 min

3.0 min

1.11

1.09

1.07

1

3

5

7

9

Fraction Number

Gradient Master profiles from 10% and 40% iodixanol at 80° and 20 rpm: effect of time

Slide 9

axis of rotation

g

At rest

Spinning

g

Swinging-bucket rotor

Slide 10

Fixed-angle

Vertical

g

1g

Fixed-angle and vertical rotors

Slide 11

Swinging-bucket

Axis of

rotation

Vertical

Sedimentation path length of rotors

Slide 12

A

B

Axis of rotation

Sample/gradient in vertical rotor

Slide 13

time

g

Self-generated gradients

  • At 360,000gav iodixanol molecules will start to sediment

Slide 14

350,000g

350,000g

1g

1g

Self-generated gradient strategy

Slide 15

Near-vertical rotors are best of all

Slide 16

Iodixanol self-generated gradient requirements

  • Rotors with a short sedimentation path length ~ 17 mm

  • RCF of 180,000-350,000gav

Slide 17

Density (g/ml)

15% iodixanol

1.30

1 h

3 h

1.25

20% iodixanol

1.20

1 h

3 h

1.15

1.10

1.05

1.00

1

3

5

7

9

11

13

15

Fraction Number

Effect of time on iodixanol self-generated gradient in the Beckman TLN100 at 365,000g

Slide 18

1.3

15% (170,000g)

30% (170,000g)

15% (353,000g)

1.2

30% (353,000g)

Density (g/ml)

1.1

1.0

1

3

5

7

9

11

Fraction Number

Effect of RCF and [iodixanol] in Beckman VTi65.1 (3 h)

Slide 19

Removal of banded material using a syringe

Slide 20

Collecting a band from a sealed tube

Slide 21

Gilson Fraction

Collector

P

96-well plate

Harvest gradient into equal volume fractions by tube puncture

Slide 22

Perfluorodecalin

Perfluorodecalin

P

P

Upward displacement

Slide 23

P

P

P

P

M

B

EP

C

1

2

3

4

Labconco Auto Densi-flow collects from meniscus

Slide 24

MB

Labconco Auto Densi-flow linked to a Gilson FC205 Fraction Collector


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